The present invention relates to calibration standards for optical illumination and detection systems, and more particularly to fluorescent standards for testing and calibrating microfluidic optical measurement systems.
Optical standards are commonly used to test and calibrate measurement systems. In general, a standard provides a benchmark that is used in a measurement system or device to maintain continuity of value in the units of measurement. In an optical measurement system, for example, an optical standard can be used to provide light having a known wavelength or intensity value to the system.
In a typical fluorescence detection system, a fluorescent material, e.g., a fluorophore, absorbs light having certain wavelengths dependent on the absorption characteristics of the material, and fluoresces (i.e., emits a fluorescent light signal) at a specific wavelength that is greater than the absorbed wavelength. In fluorescence detection systems, a fluorescence standard can be used to provide a fluorescence signal having a known wavelength and/or intensity value. Additionally, a fluorescence standard having a known fluorescent lifetime can be used.
In certain analytical detection systems, known as xe2x80x9cmicrofluidicxe2x80x9d systems, fluorescent materials are often used to measure and detect reactions and conditions. In such systems a fluorescent material is transported along a microscale channel to a detection region where the material is excited by an excitation source and the resulting fluorescent signal is measured to determine the presence or absence of some material or condition. Many such microfluidic analytical systems use a substrate or chip having a plurality of buffer and sample reservoirs interconnected by a plurality of microchannels. One or more of the microchannels typically traverse the detection region on the chip. When placed in the appropriate position relative to an illumination source and detector(s) any number of assays involving fluorescent signal detection and measurement can be performed.
To ensure continuity of value in the units of measurement, a test chip having a fluorescent material (i.e., standard) in a location corresponding to the microchannel(s) in the detection region on the assay chips can be appropriately positioned in the system to test and calibrate the illumination and detection components. In such systems, the illumination source and associated optics are designed and configured to focus the excitation radiation onto the microchannel(s) in the detection region of the assay chips. Focussing the radiation at this position provides the greatest intensity to enhance analytical detection measurements on the assay chips. In test chips, the fluorescent material is therefore located at the position corresponding to the location of the detection region. Unfortunately, however, some fluorescent materials may provide too great of a fluorescence signal to be useful when in the presence of such strong, intense excitation radiation. Additionally, some fluorescent materials may photobleach readily in such an environment thereby causing the fluorescence to be non-stable and non-constant. In some cases, photobleaching may damage the standard, requiring the expense of purchasing additional standards. It may be possible to reconfigure the illumination optics to reduce the intensity of radiation applied to the fluorescent material in such test chips, but this too requires additional expense and down time of the analytical system.
Accordingly, what is needed in the art is a fluorescent standard for use in microfluidic optical measurement systems that overcomes the above and other problems.
The present invention solves the above and other problems by providing a fluorescent standard for use in optical detection systems generally, and optimally for use in microfluidic analytical systems employing fluorescent detection techniques. The present invention provides a fluorescent standard that minimizes photobleaching of the fluorescent material and that supplies a controllably reduced fluorescent signal.
According to the present invention, a test device for use as a fluorescent standard in microfluidic analytical detection systems is provided. The test device is substantially the same size as a corresponding analysis chip for ease of use with the analytical system. The device includes one or more slits that correspond to, and are of similar dimension to, one or more microchannels in a detection region on the corresponding analysis chip. A fluorescent material is attached to a test device on the side opposite the illumination source such that excitation radiation passes through the slit(s), which defines the focal plane of the illumination optics, and impinges on the fluorescent material thereby causing the fluorescent material to fluoresce. By displacing the fluorescent material relative to the focal plane, the intensity of the radiation exciting the fluorescent material is dispersed relative to the intensity of the radiation at the focal plane, and concomitantly the strength of the resulting fluorescent signal is reduced. An optional spacer is provided to increase the distance of the fluorescent material from the focal plane so as to increase the dispersion of the radiation (decrease the intensity impinging on the fluorescent material). Such a device is useful for reducing the effect of photobleaching of the selected fluorescent material and for reducing the strength of the resulting fluorescent signal. Additionally, the strength of the resulting fluorescent signal from the fluorescent material can be controlled by selecting a spacer with the appropriate depth.
According to an aspect of the invention, a device is provided for use in testing microfluidic fluorescence detection systems having a light source and a detector for detecting fluorescent emissions. The device typically comprises a test substrate having a microslit through which light from the light source is able to pass, the microslit defining a first region, and a fluorescent material coupled to the substrate and positioned proximal the microslit, wherein the fluorescent material emits a fluorescent emissions signal when light impinges thereon, wherein when the light is focused onto the first region, at least a portion of the light passes through the microslit and is dispersed relative to the first region when it impinges on the fluorescent material. The device also typically includes a spacer coupling the substrate to the fluorescent material for increasing the amount of dispersion of the light that impinges on the fluorescent material so as to reduce even further the fluorescent signal emitted by the fluorescent material as well as the effect of photobleaching of the fluorescent material.
According to another aspect of the present invention, a device is provided for use in testing microfluidic fluorescence detection systems having a light source and a detector for detecting fluorescent emissions. The device typically comprises a test substrate having a microslit through which light from the light source is able to pass, the microslit defining a first region, and a fluorescent material coupled to the substrate and positioned proximal the microslit and opposite the light source, wherein when the light is focused onto the first region such the light has a first intensity at the first region, at least a portion of the light passes through the microslit and is dispersed such that the light has a second intensity at the fluorescent material, wherein the second intensity is lower than the first intensity, and wherein the fluorescent material emits a fluorescent emissions signal proportional to the intensity of light impinging thereon.
According to a further aspect of the present invention, a method is provided for testing a microfluidic fluorescent detection system having an excitation source and a fluorescence detector. The method typically comprises the steps of providing a test substrate coupled to a fluorescent material, the substrate having a microslit defining a first region through which light from the excitation source is able to pass through to the fluorescent material, and focusing light from the excitation source onto the first region, wherein at least a portion of the light focussed on the first region passes through the microslit so as to excite the fluorescent material. The method also typically includes the step of detecting fluorescent emissions from the excited fluorescent material with the fluorescence detector.
Reference to the remaining portions of the specification, including the drawings and claims, will realize other features and advantages of the present invention. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with respect to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements.
FIG. 1 depicts an example of a microfluidic assay device according to the present invention;
FIG. 2 is a block diagram of an exemplary microfluidic fluorescence detection system 200 according to one embodiment of the present invention;
FIG. 3 illustrates a top view of microscale test chip having a slit according to an embodiment of the present invention;
FIGS. 4a and 4b illustrate side views of the test chip of FIG. 3 coupled to a fluorescent material positioned proximal the slit according to embodiments of the present invention; and
FIG. 5 illustrates a cross section of a focussed light beam impinging on the fluorescent material through the slit according to an embodiment of the present invention.